2
04
N. Zhao et al. / International Journal of Pharmaceutics 411 (2011) 197–205
much lower and circulation times in vivo can be prolonged (Kichler,
004). By using a low molecular weight PEI coupled with disulfide
Forrest, M.L., Koerber, J.T., Pack, D.W., 2003. A degradable polyethylenimine deriva-
tive with low toxicity for highly efficient gene delivery. Bioconjug. Chem. 14,
2
934–940.
containing PEG, several advantages are achieved: (1) transfection
efficiency of low molecular weight PEI could be enhanced, (2) PEG
leads to a shielding effect of highly positive charges and therefore
enhances stability and limits cytotoxic side effects, (3) low molec-
ular weight PEI alone could be shown to be less toxic than high
molecular weight PEI, and (4) disulfide bridges lead to degradabil-
ity and with our polymer to low molecular weight PEI products
which can be more easily transported out of the cell without
accumulation.
Gao, C.X., Kim, K.-S., Liu, D., 2007. Nonviral gene delivery: what we know and what
is next. AAPS J. 9, E92–E103.
Godbey, W.T., Wu, K.K., Mikos, A.G., 1999a. Poly(ethylenimine) and its role in gene
delivery. J. Control. Release 60, 149–160.
Godbey, W.T., Wu, K.K., Mikos, A.G., 1999b. Size matters: molecular weight affects
the efficiency of poly(ethylene imine) as a gene delivery vehicle. J. Biomed.
Mater. Res. 45, 268–275.
Gosselin, M.A., Guo, W., Lee, R.J., 2001. Efficient gene transfer using reversibly
crosslinked low molecular weight polyethylenimine. Bioconjug. Chem. 12,
989–994.
Harpe, A., Petersen, v., Li, H., Kissel, Y.T., 2000. Characterization of commer-
cially available and synthesized polyethylenimines for gene delivery. J. Control.
Release 69, 309–322.
Hein, C.D., Liu, X.-M., Wang, D., 2008. Click chemistry, a powerful tool for pharma-
ceutical sciences. Pharm. Res. 25, 2216–2230.
4
. Conclusions
Jiang, X., Liu, J., Xu, L., Zhuo, R., 2011. Disulfide-containing hyperbranched
polyethylenimine derivatives via click chemistry for nonviral gene delivery.
Macromol. Chem. Phys. 212, 64–71.
Kichler, A., 2004. Gene transfer with modified polyethylenimines. J. Gene Med. 6,
S3–S10.
A high molecular weight biodegradable disulfide containing
PEG–PEI copolymer consisting of low molecular weight building
blocks was synthesized and characterized with NMR and GPC. For
block-copolymerization of PEG and PEI, click chemistry was used
and offered easier synthetic routes, high cross-linking efficiency,
milder reaction conditions, and an easier purification process,
compared with other block copolymer synthesis. Additionally,
this PEG–PEI copolymer showed a promising feature for effec-
tive gene delivery. A rapid and total degradation of the PEG–PEI
copolymer within 2 h in the presence of reducing agent was
observed. It indicated a rapid DNA releasing ability to facilitate
the following gene expression and a reduced toxicity by disul-
fide bond cleavage into small PEI-products. Gel retardation assay
demonstrated strong DNA condensation abilities of the PEG–PEI
copolymer by forming nanosized polyplexes at low N/P ratios.
The DLS and AFM measurements showed further proofs for the
small size and morphology of the resulting polyplexes. MTT-assay
indicated that the cytotoxicity of the synthesized PEG–PEI copoly-
mer is comparable with non-toxic PEI 2k and much less toxic
than PEI 25k. After coupling of small PEG chains and cross-linking
by disulfide bridges, an increased transfection efficiency in com-
parison to PEI 2k and free plasmid DNA could be obtained and
approximately 17% of the transfection efficiency of PEI 25k was
reached.
Kichler, A., Chillon, M., Leborgne, C., 2002. Intranasal gene delivery with
polyethylenimine–PEG conjugate. J. Control. Release 81, 379–388.
a
Kircheis, R., Blessing, T., Brunner, S., Wightman, L., Wagner, E., 2001. Tumor targeting
with surface-shielded ligand—polycation DNA complexes. J. Control. Release 72,
165–170.
Kleemann, E., Dailey, L.A., Abdelhady, H.G., Gessler, T., Schmehl, T., Roberts, C.J.,
Davies, M.C., Seeger, W., Kissel, T., 2004. Modified polyethylenimines as non-
viral gene delivery systems for aerosol gene therapy: investigations of the
complex structure and stability during air-jet and ultrasonic nebulization. J.
Control. Release 100, 437–450.
Kleemann, E., Neu, M., Jekel, N., Fink, L., Schmehl, T., Gessler, T., Seeger, W.,
Kissel, T., 2005. Nanocarriers for DNA delivery to the lung based upon a
TAT-derived peptide covalently coupled to PEG–PEI. J. Control. Release 109,
299–316.
Kunath, K., Harpe, A., Fischer, v., Petersen, D., Bickel, H., Voigt, U., Kissel, K.T., 2003.
Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery:
comparison of physicochemical properties, transfection efficiency and in vivo
distribution with high-molecular-weight polyethylenimine. J. Control. Release
89, 113–125.
Kursa, M., Walker, G.F., Roessler, V., Ogris, M., Roedl, W., Kircheis, R., Wagner, E.,
2
003. Novel shielded transferrin–polyethylene glycol–polyethylenimine/DNA
complexes for systemic tumor-targeted gene transfer. Bioconjug. Chem. 14,
22–231.
2
Lee, Y., Koo, H., Jin, G.-W., Mo, H., Cho, M.Y., Park, J.-Y., Choi, J.S., Park, J.S., 2005.
Poly(ethylene oxide sulfide): new poly(ethylene glycol) derivatives degradable
in reductive conditions. Biomacromolecules 6, 24–26.
Lee, Y., Mo, H., Koo, H., Park, J.-Y., Cho, M.Y., Jin, G.-W., Park, J.-S., 2007. Visualization
of the degradation of a disulfide polymer linear poly(ethylenimine sulfide), for
gene delivery. Bioconjug. Chem. 18, 13–18.
Li, J., Crasto, C.F., Weinberg, J.S., Amiji, M., Shenoy, D., Sridhar, S., Bubley, G.J., Jones,
G.B., 2005. An approach to heterobifunctional poly(ethyleneglycol) bioconju-
gates. Bioorg. Med. Chem. Lett. 15, 5558–5561.
Liu, J., Jiang, X., Xu, L., Wang, X., Hennink, W.E., Zhuo, R., 2010. Novel reduction-
responsive cross-linked polyethylenimine derivatives by click chemistry for
nonviral gene delivery. Bioconjug. Chem. 21, 1827–1835.
In summary, this disulfide containing biodegradable PEG–PEI
copolymer synthesized by click chemistry is a promising candi-
date for application in in vivo gene delivery. This method offers the
possibility of coupling LMW PEGylated PEI into HMW PEGylated
PEI containing totally biodegradable disulfide bonds. The structure
of the polymer can still be modified by changing the PEG chain
length or PEGylation degree. Further studies for improving the
pharmaceutical properties such as transfection efficiency will be
very interesting.
Lungwitz, U., Breunig, M., Blunk, T., Göpferich, A., 2005. Polyethylenimine-
based non-viral gene delivery systems. Eur. J. Pharm. Biopharm. 60, 247–
266.
Luten, J., van Nostrum, C.F., Smedt, S.C.D., Hennink, W.E., 2008. Biodegradable poly-
mers as non-viral carriers for plasmid DNA delivery. J. Control. Release 126,
97–110.
Acknowledgment
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J. Immunol. Methods 65,
55–63.
Neu, M., Fischer, D., Kissel, T., 2005. Recent advances in rational gene transfer vec-
tor design based on poly(ethylene imine) and its derivatives. J. Gene Med. 7,
The author wants to thank Thomas Betz for the AFM measure-
ments.
992–1009.
References
Neu, M., Sitterberg, J., Bakowsky, U., Kissel, T., 2006. Stabilized nanocarriers for
plasmids based upon cross-linked poly(ethylene imine). Biomacromolecules 7,
3
428–3438.
Dedola, S., Nepogodiev, S.A., Field, R.A., 2007. Recent applications of the
I
Neu, M., Germershaus, O., Behe, M., Kissel, T., 2007a. Bioreversibly crosslinked poly-
plexes of PEI and high molecular weight PEG show extended circulation times
in vivo. J. Control. Release 124, 69–80.
Neu, M., Germershaus, O., Mao, S., Voigt, K.H., Behe, M., Kissel, T., 2007b. Crosslinked
nanocarriers based upon poly(ethylene imine) for systemic plasmid delivery:
in vitro characterization and in vivo studies in mice. J. Control. Release 118,
Cu -catalysed Huisgen azide-alkyne 1,3-dipolar cycloaddition reaction in car-
bohydrate chemistry. Org. Biomol. Chem. 5, 1006–1017.
Fischer, D., Bieber, T., Li, Y., Elsässer, H., Kissel, T., 1999. A novel non-viral vector
for DNA delivery based on low molecular weight, branched polyethylenimine:
effect of molecular weight on transfection efficiency and cytotoxicity. Pharm.
Res. 16, 1273–1279.
Fischer, D., Harpe, A.v., Kissel, T., 2000. Polyethylenimine: polymer structure influ-
ences the physicochemical and biological effects of plasmid/PEI complexes. In:
Park, K.D., et al. (Eds.), Biomaterials and Drug Delivery toward New Millenium.
370–380.
Ogris, M., Brunner, S., Schuller, S., 1999. PEGylated DNA/transferring-PEI complexes:
reduced interaction with blood components, extended circulation in blood and
potential for systemic gene delivery. Gene Ther. 6, 595–605.
Peng, Q., Hu, C., Cheng, J., Zhong, Z., Zhuo, R., 2009. Influence of disulfide density and
molecular weight on disulfide cross-linked polyethylenimine as gene vectors.
Bioconjug. Chem. 20, 340–346.
,
pp. S195–S211.
Fischer, D., Li, Y., Ahlemeyer, B., Krieglstein, J., Kissel, T., 2003. In vitro cytotoxic-
ity testing of polycations: influence of polymer structure on cell viability and
hemolysis. Biomaterials 24, 1121–1131.